1. Technical Field
The present invention relates to a yeast extract containing γ-Glu-X-Gly or γ-Glu-X and a method for producing the same. The yeast extract of the present invention is useful in the field of foodstuffs such as seasonings and health foods.
2. Background Art
Yeast extracts have a function of imparting atsumi (thickness), umami, etc. to foodstuffs, and have been widely used as seasonings in the field of foodstuffs. Especially, glutathione (henceforth also referred to as “GSH”), which is a tripeptide consisting of glutamic acid, cysteine and glycine, is known to impart kokumi to foodstuffs (Ueda et al., Agric. Biol. Chem., 54, 163-169 (1990), Ueda et al., Biosci. Biotechnolo. Biochem., 61, 1977-1980 (1997)), and seasonings containing GSH have been developed.
Meanwhile, although the calcium sensing receptor (CaSR), which is a G-protein classified into the class C, has been reported to respond to GSH (Wang et al., Journal of Biological Chemistry, 281, 8864-8870 (2006)), the physiological significance thereof has not been clarified. Moreover, this CaSR is present also in the lingual cells, and it was considered to show a certain taste response (Gabriel et al., Biochemical and Biophysical Research Communications, 378, 414-418 (2009)). Then, it has recently been clarified that this CaSR participates in recognition of kokumi in humans (Ohsu et al., Journal of Biological Chemistry, 285, 1016-1022 (2010)). This reference reported that not only GSH has been recognized as a kokumi substance, but also several γ-glutamyl compounds similarly respond to CaSR. Furthermore, it has been reported that peptides represented by the general formula γ-Glu-X or γ-Glu-X-Gly (X can represent an amino acid or amino acid derivative other than Cys), for example, γ-Glu-Met, γ-Glu-Thr, γ-Glu-Val-Gly, etc. have a kokumi-imparting effect (WO2007/055393). Moreover, the group of esters including S- or O-carboxyalkylated γ-glutamyl or β-asparagyl peptides etc., are also reported as kokumi substances (WO2007/042288). Although these peptides impart kokumi to foodstuffs like GSH, they do not have a reduced SH group unlike GSH. It is known that a substance having the reduced SH group such as GSH is generally unstable, and titer thereof is reduced with formation of disulfide bond (WO2007/042288). However, γ-Glu-X, γ-Glu-X-Gly etc. are considered useful from the viewpoint that the kokumi-imparting peptides not having the reduced SH group are stable.
As for foodstuffs containing a γ-Glu dipeptide, it has been reported that various γ-Glu dipeptides were detected in Gouda cheese ripened over a long period of time, even as long as about 44 weeks (Toelstede, S and Hofmann, T, J. Agric. Food. Chem., 2009). This reference reported that various γ-Glu dipeptides such as γ-Glu-Ala, γ-Glu-Glu and γ-Glu-Gln were detected in a total amount of 3590 μmol/kg of dry materials at most. This value corresponds to 0.088% in terms of weight percentage based on the solid content.
However, a yeast extract containing γ-Glu-X and γ-Glu-X-Gly in an amount enabling impartation of kokumi has not been previously reported.
Furthermore, it is known that the synthesis and decomposition of glutathione, which is one of the γ-glutamyl compounds, is catalyzed by a plurality of enzymes which make up the γ-glutamyl cycle. In particular, γ-glutamyl transpeptidase is known to transfer the glutamate of GSH at the γ-position to another compound having an amino group, resulting in decomposition of GSH to cysteinylglycine (Protein Nucleic acid Enzyme, 1988-7, VOL. 33, NO. 9, ISSN 003909450, Special Issue “Epoch of glutathione research”, pp. 1432-1433). It is considered that, if the compound having an amino group in this rearrangement reaction is an amino acid, a dipeptide of γ-Glu-X can be generated as a by-product. However, research into making a microorganism effectively produce this by-product has not been positively performed to date, partially because it is a by-product.
Findings about the dipeptide γ-Glu-X that have been reported include an analysis of the fermentation broth of Micrococcus glutamicus (Ronald et al., Journal of Biological Chemistry, 240, p 2508-2511 (1965)). This reference reported that the fermentation broth was loaded onto various columns to separate peptides etc., and to isolate γ-Glu-Glu, γ-Glu-Val, and γ-Glu-Leu. However, these were found as a result of separation with various columns, and the amounts of these peptides contained in the broth were not determined.
Furthermore, an enzyme responsible for GSH biosynthesis was newly isolated from Streptococcus agalactiae and Clostridium acetobutylicum, and the substrate specificity thereof was analyzed (Kino et al., JBB research communications, 352, pp. 351-359 (2007)). GSH is usually biosynthesized by two different enzymes called γ-glutamylcysteine synthetase, which combines Glu and Cys to generate γ-Glu-Cys, and glutathione synthetase, which combines the produced γ-Glu-Cys and Gly to generate GSH. However, the aforementioned two kinds of microorganisms have a unique enzyme which is essentially a fusion of γ-glutamylcysteine synthetase and glutathione synthetase. It was reported that, according to an in vitro analysis, the substrate recognition of this enzyme was slightly ambiguous, i.e., it also recognized amino acids other than Cys, and as a result, it could generate γ-Glu-X and γ-Glu-X-Gly. However, these are nevertheless in vitro results, and it was not described whether or not those microorganisms that produced marked amounts of peptides such as γ-Glu-X and γ-Glu-X-Gly, also contain many compounds having an amino group besides the target X.
Yeast extracts are seasonings which have been widely used in the field of foodstuffs, and are highly accepted by consumers. Therefore, a yeast extract can be used as a carrier of γ-Glu-X-Gly or γ-Glu-X. Yeast strains containing minerals have been studied for this use. It is known that if a metal is added to a medium, yeasts take up the metal into the cells (B. Volesky, H. A., Appl. Microbiol. Biotechnol., 42; 797-806 (1995)). In particular, if trace elements such as zinc, iron, copper, manganese, selenium, molybdenum and chromium are added to the medium, yeasts can be used to supply such trace elements via enrichment in foodstuffs (Japanese Patent Laid-open (Kokai) No. 2004-298014). As a result, methods for producing mineral-containing yeast have been developed (Japanese Patent Laid-open No. 54-157890, Japanese Patent Laid-open No. 60-75279, Japanese Patent Publication (Kokoku) No. 6-16702).
Furthermore, mineral-containing yeast also have an advantage concerning taste. For example, yeast containing a high amount of magnesium are described in Japanese Patent Laid-open No. 8-332081. This reference describes that although magnesium-enriched foodstuffs containing inorganic magnesium salt were also marketed, a strong bitterness and astringency was noted due to the mineral salt. As a result, it was quite more difficult to routinely eat the magnesium-enriched foodstuffs containing inorganic magnesium salt as compared to foodstuffs containing naturally occurring magnesium. Japanese Patent Laid-open No. 8-332081 also discloses a technique of producing a natural material by making yeast take up magnesium. As for nutritional merit, the technique disclosed in Japanese Patent Laid-open No. 2008-99578 can be exemplified. According to this reference, although zinc contributes to improving taste and generative function, etc., the reference overlooks that zinc is often not taken in sufficient amounts. If zinc is added during the yeast cultivation process, yeast takes up zinc into cells, but water-soluble zinc binds with a protein or an amino acid, and accumulates as amorphous zinc at a high concentration. The amorphous zinc is more efficiently absorbed into the body, as compared to crystalline zinc. As a result, improved absorption can be obtained by incorporating zinc into yeast, as compared to simply taking zinc as it is.
As described above, there are various advantages to making yeast take up a target substance and adding either the yeast or a yeast extract to foodstuffs, as compared to simply adding the target substance to foodstuffs. However, unlike minerals, which are essential nutrients, the ability of yeast to take up an amino acid or a peptide is delicately controlled, and it simply applying the technique for incorporating minerals into yeast to the techniques for uptake of amino acid or peptides was considered to be difficult.
There has been much research concerning the generation of GSH or γ-Glu-Cys using yeast Examples of such research include the report that the GSH content was improved by mutagenizing a Saccharomyces yeast and selecting a strain having improved zinc resistance (Japanese Patent Laid-open No. 02-295480), the report that suppression of the MET25 gene expression was derepressed by making a cell contain a mutant MET30 gene and thereby increase intracellular γ-Glu-Cys content (Japanese Patent Application No. 2002-282743), and so forth. Moreover, the latest scientific findings include uptake of GSH by Hgt1p. Although GSH and the dimer thereof, GSSG, were taken into cells by Hgt1p, uptake of GSH by Hgt1p was not affected even in the presence of excessive amounts of amino acids, various dipeptides, and tripeptides. Therefore, it is considered that Hgt1p is not a nonspecific transporter as once thought, but a transporter specific to GSH (Bourbouloux et al., Journal of Biological Chemistry, 275, pp. 13259-13265 (2000)). Furthermore, a search for the active site of Hgt1p has also been performed (Kaur et al., FEMS Yeast Res., 9, 849-866 (2009)).
As described above, although many findings about GSH and the precursor thereof, γ-Glu-Cys, have been reported, there have been no reports about yeast cells containing such a substance as γ-Glu-X and γ-Glu-X-Gly, and a method for producing an extract prepared from the cells.